Transducin, the major guanine nucleotide binding protein in vertebrate retina functions to couple the receptor, rhodopsin, to effector, CGMP- phosphodiesterase. Like other heterotrimeric G proteins, transducin consists of alpha, beta, and gamma subunits. The alpha subunit of transducin is cotranslationally modified at the amino terminus with a myristoyl or structurally related N-acyl group. The amino terminus of Gtalpha also interacts with the Gtbeta gamma subunit. Two monoclonal antibodies, LAS1 and LAS2, made against purified bovine Gtalpha were mapped with high precision to the amino terminus of Gtalpha by proteolytic modification and recombinant methodologies. Removal of a 2- Kda fragment from the amino terminus of the 39 Kda Gtalpha by trypsinization abolished reactivity with both antibodies, of the remaining 37 Kda product on Western Blots. When subjected to arginine-C proteolytic cleavage, reactive products were found at 34 Kda, 23 Kda and 15 Kda, none of which could be sequenced by Edman degradation, consistent with the hypothesis that these fragments contained a blocked amino terminus. The amino terminal modification with an acyl group is required for antibody recognition as expression of Gtalpha in E. coli without co- expression of an N-myristoyltransferase resulted in a recombinant Gtalpha that did not react with either monoclonal antibody, however, when coexpressed in E. coli with N-myristoyltransferase and myristoylated, reactivity of the myristoylated Gtalpha with the antibodies was seen. Although by all physical mapping studies the epitopes of these antibodies appear to be identical, functionally, the antibodies differ in inhibition of the Gtbeta gamma-dependent pertussis toxin-catalyzed ADP-ribosylation of Gtalpha as LAS2, but not LAS1 inhibited this reaction. Based on these studies, it appears that LAS 1 and LAS 2 recognize a fatty acylated amino terminus but differ in their binding properties.